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PubMed1997

A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains.

Pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains are structurally related regulatory modules that are present in a variety of proteins involved in signal transduction, such as kinases, phospholipases, GTP exchange proteins, and adapter proteins. Initially these domains were shown to mediate protein-protein interactions, but more recently they were also found to bind phosphoinositides. Most studies to date have focused on binding of PH domains to phosphatidylinositol (PtdIns)-4-P and PtdIns-4,5-P2 and have not considered the lipid products of phosphoinositide 3-kinase: PtdIns-3-P, PtdIns-3,4-P2, and PtdIns-3,4,5-P3. Here we have compared the phosphoinositide specificity of six different PH domains and the Shc PTB domain using all five phosphoinositides. We show that the Bruton's tyrosine kinase PH domain binds to PtdIns-3,4, 5-P3 with higher affinity than to PtdIns-4,5-P2, PtdIns-3,4-P2 or inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4). This selectivity is decreased by the xid mutation (R28C). Selective binding of PtdIns-3,4,5-P3 over PtdIns-4,5-P2 or PtdIns-3,4-P2 was also observed for the amino-terminal PH domain of T lymphoma invasion and metastasis protein (Tiam-1), the PH domains of Son-of-sevenless (Sos) and, to a lesser extent, the PH domain of the beta-adrenergic receptor kinase. The oxysterol binding protein and beta-spectrin PH domains bound PtdIns-3,4,5-P3 and PtdIns-4,5-P2 with similar affinities. PtdIns-3,4,5-P3 and PtdIns-4,5-P2 also bound to the PTB domain of Shc with similar affinities and lipid binding was competed with phosphotyrosine (Tyr(P)-containing peptides. These results indicate that distinct PH domains select for different phosphoinositides.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2002

Association of Grb7 with phosphoinositides and its role in the regulation of cell migration.

Grb7 is the prototype of a family of adaptor molecules that also include Grb10 and Grb14 that share a conserved molecular architecture including Src homology 2 (SH2) and pleckstrin homology (PH) domains. Grb7 has been implicated as a downstream mediator of integrin-FAK signal pathways in the regulation of cell migration, although the molecular mechanisms are still not well understood. In this paper, we investigated the potential role and mechanisms of PH domain in Grb7 in the regulation of cell migration. We found that the PH domain mediated Grb7 binding to phospholipids both in vitro and in intact cells. Furthermore, both Grb7 and its PH domain preferentially interacted with phosphatidylinositol phosphates showing strongest affinity to the D3- and D5-phosphoinositides. The PH domain interaction with phosphoinositides was shown to play a role in the stimulation of cell migration by Grb7. It was also shown to be necessary for Grb7 phosphorylation by FAK, although it was not required for Grb7 interaction with FAK or recruitment to the focal contacts. Last, we found that PI 3-kinase activity played a role in both Grb7 association with phosphoinositides and its stimulation of cell migration. In addition, both FAK binding to PI 3-kinase via its autophosphorylated Tyr(397) and integrin-mediated cell adhesion increased Grb7 association with phosphoinositides. Together, these results identified the Grb7 PH domain interaction with phosphoinositides and suggested a potential mechanism by which several signaling molecules including Grb7, FAK, and PI 3-kinase and their interactions cooperate to mediate signal transduction pathways in integrin-mediated cell migration.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Rho GTPases and phosphoinositide 3-kinase organize formation of branched dendrites.

Neurons receive information from other neurons via their dendritic tree. Dendrites and their branches result from alternating outgrowth and retraction. The Rho GTPases Rac and Cdc42 (cell division cycle 42) facilitate the outgrowth of branches, whereas Rho attenuates it. The mechanism of neurite retraction is unknown. Because the adenylyl cyclase activator forskolin causes numerous branched extensions in NG108-15 cells, we have investigated the underlying mechanism in this cell line. In additional studies, we used cultured hippocampal neurons in which forskolin induces branched dendrites. In both cell types, forskolin enhanced the activity of Cdc42, but not that of Rac, although both GTPases were necessary for the formation of branched extensions. Time lapse microscopy showed that forskolin did not increase the rate of addition of new extensions or branches, but it reduced the rate of the retraction so that more branched extensions persisted. Inhibition of phosphoinositide 3-kinase activity by wortmannin or LY294002 also reduced the rate of retraction and thus facilitated dendritic arborization. Forskolin diminished the activity of phosphoinositide 3-kinases. Inhibitors of phosphoinositide 3-kinases not only reduced the retraction but also the addition of new dendrites and branches. This reduction was no longer present when Rho kinase was simultaneously inactivated, suggesting an interaction of phosphoinositide 3-kinases and Rho kinase. The present results show a central role of phosphoinositide 3-kinases in dendrite formation. In neuronal cells, increased levels of cAMP can support dendritic arborization by modulating the activity of the lipid kinase.

Amides↗

PubMed2003

Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain.

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP.

Amino Acid Sequence↗

PubMedDate not supplied

Membrane targeting and remodeling through phosphoinositide-binding domains.

In mammals, there are seven inositolphospholipids, collectively called phosphoinositides that serve as versatile molecules not only in receptor-mediated signal transduction but also in a variety of cellular events such as cytoskeletal reorganization, membrane trafficking, cell proliferation and cell death. Recent studies have revealed that the latter functions are mediated by direct interactions between phosphoinositides and proteins. Such proteins contain two types of phosphoinositide-binding regions; basic amino acid stretch and globular structural domain. Furthermore, spatially restricted compartment of phosphoinositides and their concentration are finely regulated by a large number of phosphoinositide kinases and -phosphatases, controlling localization-specific metabolism of this simple lipid whose aberrations cause various diseases such as cancer and diabetes.

Animals↗

PubMed1984

Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine.

The absorption of light by photoreceptor cells leads to an increased incorporation of [2-3H]inositol into phosphoinositides of horizontal cells in the retina of Xenopus laevis in vitro. We have identified several retinal neurotransmitters that are involved in regulating this response. Incubation with glycine, the neurotransmitter of an interplexiform cell that has direct synaptic input onto horizontal cells, abolishes the light effect. This inhibition is reversed by preincubation with strychnine. Acetylcholine added to the culture medium enhances the incorporation of [2-3H]inositol into phosphoinositides in horizontal cells when retinas are incubated in the dark. This effect is inhibited by preincubation with atropine. However, atropine alone does not inhibit the light-enhanced incorporation of [2-3H]inositol into phosphoinositides in the retina. gamma-Aminobutyric acid, the neurotransmitter of retinal horizontal cells in X. laevis, as well as dopamine and norepinephrine, have no effect on the incorporation of [2-3H]inositol into phosphoinositides. These studies demonstrate that the light-enhanced incorporation of [2-3H]inositol into phosphoinositides of retinal horizontal cells is regulated by specific neurotransmitters, and that there are probably several synaptic inputs into horizontal cells which control this process.

Acetylcholine↗

PubMed1995

Veratrine-stimulated phosphoinositide breakdown as an assay for local anesthetic actions at Na+ channels.

The blockade of veratrine-stimulated phosphoinositide breakdown in rat cerebral cortical miniprisms as a model of local anesthetic actions on voltage-dependent sodium channels was assessed. Veratrine stimulated phosphoinositide breakdown with an EC50 value of 5 microM. The stimulation produced by 20 microM veratrine was blocked completely by (+)-bupivacaine (IC50 7.6 microM [mean of three separate experimental series]), (-)-bupivacaine (IC50 7.3 microM), lidocaine (IC50 34 microM), etidocaine (IC50 3.4 microM), tetracaine (IC50 approximately 2 microM), and prilocaine (IC50 110 microM). Phosphoinositide breakdown responses to ouabain (100-1000 microM) and K+ (50 mM) were only partially blocked by (+)-bupivacaine, and the responses to monensin (100 and 1000 microM) and noradrenaline (30 microM) were not blocked at all by this drug. Nifedipine produced no significant effects on the phosphoinositide response to 10 microM veratrine. It is concluded that in pulse label experiments using rat cerebral cortical miniprisms, local anesthetics in general, and (+)-bupivacaine in particular, block the phosphoinositide response to veratrine with a high degree of specificity. This system may be useful as a relatively simple and quantitative assay for drug effects on Na(+)-channels.

Anesthetics, Local↗

PubMed1986

Agonist-induced phosphoinositide hydrolysis in choroid plexus.

5-Hydroxytryptamine (5-HT, serotonin) stimulates phosphoinositide hydrolysis in choroid plexus by interacting with the 5-HTlc site. In the present study, the effects of 5-HT were compared with those of other agonists. 5-HT stimulates a rapid release of all three inositol sugars in a mianserin-sensitive manner. Inositol bisphosphate and inositol trisphosphate levels increase about twofold within 2.5 min, whereas inositol monophosphate levels are not appreciably elevated until 5 min. In contrast, glutamate, carbachol, histamine, substance P, and vasopressin, agents that increase phosphoinositide hydrolysis in other tissues, do not stimulate this response in choroid plexus. High concentrations of norepinephrine increase inositol phosphate release in choroid plexus, but this effect is apparently mediated by activation of the 5-HTlc site. The depolarizing agents KCl and veratrine also fail to stimulate phosphoinositide hydrolysis in choroid plexus. These results, combined with the finding that the phosphoinositide response to 5-HT is insensitive to tetrodotoxin, suggest that the effects of 5-HT are not secondary to neurotransmitter release. Furthermore, an indirect effect mediated via arachidonic acid metabolism is unlikely, since inhibitors of cyclooxygenase and lipoxygenase do not reduce the 5-HT response. We conclude, therefore, that phosphoinositide hydrolysis is the transducing mechanism of the 5-HT 5-HTlc receptor and that the choroid plexus will serve as a useful model system for studies of this receptor.

Animals↗

PubMed1990

Exogenous gangliosides stimulate breakdown of neuro-2A phosphoinositides in a manner unrelated to neurite outgrowth.

Gangliosides administered exogenously are well-known effectors of differentiation in many neuroblastoma lines and primary neuronal cultures. Previous studies suggested the phosphoinositide signaling mechanism could be a contributing factor. We have found that treatment of Neuro-2A cells with bovine brain ganglioside mixture (BBG) causes breakdown of phosphoinositides, as measured by increased levels of inositol phosphates. The effect was optimal at 60 min and required a minimal BBG concentration of 25 microM. However, addition of neomycin, which blocked phosphoinositide breakdown, had no observable effect on ganglioside-stimulated neurite outgrowth. A similar result was obtained with psi-tectorigenin, which also inhibited phosphoinositide hydrolysis. When cells were treated with maitotoxin, an agent that promotes phosphoinositide breakdown, there was no enhancement of neurite outgrowth. These findings indicate that although exogenous gangliosides elevate inositol phosphate formation over a prolonged period in neuro-2A cells, this reaction is not integral to the differentiation of these cells. The possibility of secondary effects influencing neurite type and structure cannot be excluded.

Adenosine Triphosphate↗

PubMed1995

Role of glucose metabolism and phosphoinositide hydrolysis in glucose-induced sensitization/desensitization of insulin secretion from mouse pancreatic islets.

The role of glucose metabolism and phosphoinositide hydrolysis in glucose-induced sensitization/desensitization of insulin secretion was studied. A change in glucose concentration from 5.5 to 16.7 mM during 22-24 h of pre-exposure of mouse islets in TCM 199 culture medium (0.26 mM Ca2+) led to sensitization of glucose-induced insulin secretion. This change in islet responsiveness to glucose was not mediated by increases in glucose utilization ([5-3H]glucose conversion to 3H2O) and glucose oxidation ([U-14C]glucose oxidation to 14CO2). Glucose-induced sensitization of insulin secretion was associated with an increase in glucose-induced phosphoinositide hydrolysis, leading to a significant increase in inositol 1-monophosphate formation, but not in inositol 1,4-bisphosphate or in inositol 1,4,5-trisphosphate plus inositol 1,3,4-trisphosphate formation. Diacylglycerol, which may arise from both phosphoinositide hydrolysis and de novo from glucose metabolism, was, on the other hand, not increased during acute exposure to glucose and not changed after pre-exposure to glucose. At 16.7 mM glucose in TCM 199 medium, a change in Ca2+ concentration from 0.26 to 1.26 mM led to a reduction in glucose-induced insulin secretion. This Ca(2+)-dependent desensitization of insulin secretion in the presence of glucose was associated with a decrease in glucose-induced phosphoinositide hydrolysis, but not with a change in glucose metabolism or diacylglycerol accumulation. In conclusion, it is suggested that glucose-induced sensitization/desensitization of insulin secretion may involve changes in phosphoinositide hydrolysis, but may occur independently of concomitant changes in glucose metabolism or diacylglycerol accumulation.

Animals↗

PubMed2006

Stress-ING out: phosphoinositides mediate the cellular stress response.

Phosphoinositides regulate numerous cellular processes required for growth, proliferation, and motility. Whereas phosphoinositide signal transduction pathways within the cytosol have been well characterized, nuclear signaling pathways remain poorly understood. Accumulating experimental data have now started to uncover critical functions for nuclear phosphoinositides. In particular, phosphoinositides modulate the activity of the tumor suppressor protein ING2 in response to extracellular stress. These findings highlight a previously uncharacterized function for phosphoinositides and implicate their metabolism in signaling pathways critical for cell survival.

Animals↗

PubMed1998

Phosphoinositide kinases.

Phosphatidylinositol, a component of eukaryotic cell membranes, is unique among phospholipids in that its head group can be phosphorylated at multiple free hydroxyls. Several phosphorylated derivatives of phosphatidylinositol, collectively termed phosphoinositides, have been identified in eukaryotic cells from yeast to mammals. Phosphoinositides are involved in the regulation of diverse cellular processes, including proliferation, survival, cytoskeletal organization, vesicle trafficking, glucose transport, and platelet function. The enzymes that phosphorylate phosphatidylinositol and its derivatives are termed phosphoinositide kinases. Recent advances have challenged previous hypotheses about the substrate selectivity of different phosphoinositide kinase families. Here we re-examine the pathways of phosphoinositide synthesis and the enzymes involved.

1-Phosphatidylinositol 4-Kinase↗

PubMed2004

Phosphoinositides in constitutive membrane traffic.

Proteins that make, consume, and bind to phosphoinositides are important for constitutive membrane traffic. Different phosphoinositides are concentrated in different parts of the central vacuolar pathway, with phosphatidylinositol 4-phosphate predominate on Golgi, phosphatidylinositol 4,5-bisphosphate predominate at the plasma membrane, phosphatidylinositol 3-phosphate the major phosphoinositide on early endosomes, and phosphatidylinositol 3,5-bisphosphate found on late endocytic organelles. This spatial segregation may be the mechanism by which the direction of membrane traffic is controlled. Phosphoinositides increase the affinity of membranes for peripheral membrane proteins that function for sorting protein cargo or for the docking and fusion of transport vesicles. This implies that constitutive membrane traffic may be regulated by the mechanisms that control the activity of the enzymes that produce and consume phosphoinositides. Although the lipid kinases and phosphatases that function in constitutive membrane traffic are beginning to be identified, their regulation is poorly understood.

Animals↗

PubMed1991

Angiotensin-induced desensitization of the phosphoinositide pathway in cardiac cells occurs at the level of the receptor.

Previous studies show that angiotensin II (Ang II) increases phosphoinositide turnover in cultured neonatal heart cells. Ang II has also been shown to transiently increase spontaneous beating behavior in these cells. In this study we seek to identify the molecular mechanism underlying this rapid (3-5-minute) desensitization. Time-course studies on the accumulation of [3H]inositol phosphates indicate that the loss in functional responsiveness correlates with reduced efficacy of Ang II to activate the phosphoinositide path. Binding studies with 125I-Ang II revealed that there was no change in surface receptor binding capacity during the time in which desensitization developed. Normal phosphoinositide and functional responses are observed when desensitized cells are treated with probes that activate the cardiac phosphoinositide pathway at discrete steps. These studies reveal that the functional status of the major components of the phosphoinositide signaling pathway, including G proteins, phospholipase C, and protein kinase C (PKC), are normal during maintained Ang II desensitization. To study the potential role of PKC in Ang II desensitization, the cells are treated with TPA for 24 hours, which downregulates PKC activity. PKC-depleted cells rapidly desensitize after Ang II application. We conclude that the selective Ang II-evoked biochemical/functional desensitization involves inhibition at the level of the receptor, rather than at a component downstream in the path, and that this process is independent of PKC and loss of surface binding capacity.

Angiotensin II↗

PubMed1988

Attenuation of anterior pituitary phosphoinositide phosphorylase activity by the D2 dopamine receptor.

We have examined the influences of dopamine and the D2 receptor agonist bromocriptine on phosphoinositide metabolism in primary cultures of rat anterior pituitary cells, monitoring changes in the levels of phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate [PtdIns(4)P], and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Basal incorporation of [3H]inositol ([3H]Ins) into phosphoinositides was progressive, and radioisotopic equilibrium was attained in all three species within 48 h. The inclusion of dopamine or bromocriptine in the incubation medium promoted concentration-dependent reductions in the rate, but not the magnitude, of phosphoinositide radiolabeling. The onset of this effect was rapid; inhibition of [3H]Ins incorporation by dopamine (500 nM) and bromocriptine (100 nM) could be detected within 2 h. This treatment also produced a comparable reduction in the incorporation of [32P]orthophosphate into PtdIns(4,5)P2. In extended time-course studies, bromocriptine dramatically retarded the radiolabeling of PtdIns(4)P and PtdIns(4,5)P2, and apparent equilibria in these species were attained only after 96 h. We also assessed the ability of dopamine to modify the concentration-response characteristics of [3H]Ins-labeled inositol phosphate ([3H]InsPx) production by TRH, angiotensin II (AII), neurotensin (NTS), bombesin (BBS), and vasoactive intestinal polypeptide (VIP). Neither dopamine nor bromocriptine altered the rate or magnitude of TRH-, AII-, NTS-, or BBS-related InsPx generation. VIP was completely ineffective in stimulating InsPx generation. PRL release was significantly reduced in all dopamine-treated groups. That the InsPx concentration-response relationships for each of these peptides remained unimpaired by exposure to dopamine or bromocriptine extends our previous observation that the phosphoinositide-specific phospholipase-C is insensitive to dopaminergic tone. Consistent with our earlier findings, these data indicate that activation of the D2 dopamine receptor attenuates the activity of mechanisms associated with the serial phosphorylations of PtdIns and PtdIns(4)P, reactions that give rise to PtdIns(4)P and PtdIns(4,5)P2, respectively. It is our conclusion that dopamine, in addition to its other actions, attenuates the phosphorylation, rather than the hydrolysis, of anterior pituitary phosphoinositide. This attenuation appears to be mediated by an inhibitory coupling of the D2 receptor with the phosphoryltransferase activities that catalyze PtdIns(4)P and PtdIns(4,5)P2 formation.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Phosphatidylinositol 4-Kinase↗

PubMed1999

Maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in human astrocytoma cells.

Since maitotoxin, a potent marine toxin, is known to cause not only Ca2+ influx but also phosphoinositide hydrolysis, we investigated the Ca2+ dependency of maitotoxin-induced phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Maitotoxin elicited inositol 1,4,5-trisphosphate accumulation in a time-dependent manner. In [3H]inositol-labeled cells, maitotoxin stimulated phosphoinositide hydrolysis in an extracellular Ca2+-dependent manner. Maitotoxin also caused an intracellular Ca2+ elevation, which was abolished by an intracellular Ca2+ chelater BAPTA-AM. Interestingly, maitotoxin still caused phosphoinositide hydrolysis in the BAPTA-AM-treated cells. These results indicate that maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in 1321N1 human astrocytoma cells.

Astrocytoma↗

PubMed2004

Measurement and immunofluorescence of cellular phosphoinositides.

Phosphoinositides are a vitally important class of intracellular-signaling molecules that regulate cellular processes, including signaling through cell-surface receptors, remodeling of the cytoskeleton, vesicle-mediated protein trafficking, and various nuclear functions. Methods for the analysis of in vivo phosphoinositide concentration, such as the one described in this chapter enable quantification of all phosphoinositides from a population of cells. This method involves metabolic labeling of cells with myo<-[2-3H] inositol, followed by lipid extraction, and quantification by high-performance liquid chromatography (HPLC). It provides improved efficiency and reproducibility when analyzing yeast, plant cells, and is applicable to animal cells as well. In addition, a technique for determining the intracellular location of phosphoinositides is described. When quantification and localization techniques are used in parallel, an investigator can identify cell, and even subcellular concentration changes. The technique described in this chapter uses immunodetection with antiphosphoinositide antibodies to determine the localization and relative concentrations of phosphinositides in fixed cells. The availability of antibodies allows an investigator to perform immunofluorescence and potentially immunoelectron microscopy of phosphoinositide localization on particular cellular, organellar, or vesicular membranes.

3T3 Cells↗

PubMed2006

A directly labeled TR-FRET assay for monitoring phosphoinositide-3-kinase activity.

Phosphoinositide 3-kinases (PI3Ks) comprise a family of kinases that transfer the terminal phosphate of adenosine triphosphate to phosphoinositides at the 3-hydroxyl of the inositol ring to form phosphoinositide (3,4,5) triphosphate (PIP3). The PI3Ks have been shown to play key roles in cell growth, motility, morphology, and survival and thus are of interest as targets in anti-inflammatory and anti-oncogenic drug development. To facilitate identification of novel and selective inhibitors of PI3Ks, we have developed a TR-FRET assay that uses directly labeled reagents. The assay makes use of the high affinity binding of phosphoinositides to a Pleckstrin homology (PH) domain in the general receptor for phosphoinositides 1 (Grp1) protein. It monitors PIP3 produced from the enzymatic reaction by measuring its competition with Bodipy-FL-labeled PIP3 for binding to Terbium chelate-labeled Grp1. By using directly labeled reagents, this assay configuration offers higher sensitivity and faster binding/dissociation kinetics than existing non-radioactive assays, which are critical for competitive assay formats. The assay is homogenous, robust (Z' = 0.88), and simple and, thus, compatible with high throughput screening (HTS) processes.

Biological Assay↗